Characterization of taxon-specific endogenous reference genes in LM soybeans and maize using chamber digital PCR and real-time PCR

The quantitative analysis of the Living Modified (LM) crops is expressed as the percentage ratio of the introduced gene copy number to that of the endogenous reference gene (ERG), highlighting the necessity of developing and validating reliable reference genes.This study evaluated the characteristics of key ERG assays used for approved LM soybean and maize events in Korea through traditional PCR, chamber digital PCR (cdPCR), and realtime quantitative PCR (RT-qPCR).For soybeans, assays targeting different regions of the lectin gene, Le1-A and Le1-B, were performed.For maize, assays targeting the high mobility group a (HMG), alcohol dehydrogenase 1 (ADH1), and fructose bisphosphate Aldolase (ALD) genes were conducted.Traditional PCR analysis was used to assess taxon specificity, while cdPCR was used to determine the target copy number and zygosity ratio.RT-qPCR was employed to analyze the slope, R2, and efficiency of the standard quantification curve.Each assay showed species specificity.The Le1-A and Le1-B assays were suitable for all tested LM soybean events and domestic varieties.For maize, the HMG and ADH1 assays were suitable for 11 and 10 of the 12 LM maize events, respectively, in addition to domestic varieties.However, the ALD assay for maize formed two distinct "positive" groups in cdPCR due to single nucleotide polymorphisms in homologous genes, suggesting its unsuitability as an ERG for maize.The ERG assays proposed in this study for LM soybean and LM maize, respectively, enable the establishment of efficient qualitative and quantitative analysis methods, potentially enhancing compliance with regulatory requirements.